A mechanical circuit in End4p coordinates force transmission during clathrin-mediated endocytosis

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Mechanical forces are transmitted from the actin cytoskeleton to the membrane during clathrin-mediated endocytosis (CME) in the fission yeast Schizosaccharomyces pombe . The onset and termination of force transmission is tightly regulated temporally during different stages of CME, and spatially over the surface of the invaginated membrane. How force transmission is regulated and coordinated at the molecular scale is unclear. An adaptor protein in CME, End4p, directly transmits force by binding to both the membrane (through ANTH domain) and F-actin (through THATCH domain). We show that 8pN is required for stable binding between THATCH and F-actin. We also report the discovery and characterization of a new domain on End4p, which we named Rend ( R domain in End 4p), that resembles R12 of talin. Membrane localization of Rend primes the binding of THATCH to F-actin, and force-induced unfolding of Rend at 15pN terminates the transmission of force during CME. We show that the mechanical properties (mechanical stability, unfolding length, hysteresis) of Rend and THATCH are tuned to form an auto-regulated circuit for the initiation, transmission and termination of force between the actin cytoskeleton and membrane. Shorting the circuit leads to permanent End4p association with the membrane or with F-actin, or failure to enter the force transmission cycle. Mathematical modeling of force transmission through Rend-THATCH connection shows that input force from F-actin is buffered to a narrow range towards the membrane. The mechanical circuit by Rend and THATCH may be conserved and coopted evolutionarily in cell adhesion complexes.

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  1. THATCH dimers really seem to be diffusing throughout the cytoplasm and the nucleus with no apparent localization to the actin patches or cables (Fig. 2b). This is consistent with our previous findings (ref. 44, 45). We attribute the residual co-localization of end4(1-856) and THATCH to incomplete cleavage by the 2A peptide. The localization of THATCH in ref. 44 and 45 is visualized at the endogenous expression level.

  2. solated THATCH dimers displayed a diffusive localization throughout the fission yeast cell, indicating a low affinity to F-actin, similarly to THATCH homologues (Fig. 2b)(44, 45). In contrast, overexpression of THATCH-ΔUSH dimers induced the formation of thick F-actin bundles and large puncta (Fig. 2c)(34)

    Very Interesting! Did you ever notice THATCH dimers associated at cables when expressed at endogenous levels? Would this indicate something in the THATCH domain is contributing to patch localization? Do the (1-856) and THATCH fragments co-localize? If so, could the 2 fragments be re-associating (infrequently)?